PROJECT SUMMARY The current Zika virus outbreak is a growing risk to public health due to the increasing association of the virus with serious secondary diseases. Research on Zika virus is crucial to help prevent the spread of the epidemic and will ideally result in the creation of therapeutic antibodies and vaccines. However, vaccine development can be complicated as some regions of viruses can rapidly mutate to evade antibody responses, while other locations are functionally conserved and thus cannot tolerate sequence changes. Targeting such conserved regions can promote the development of antibodies that are broadly neutralizing. We hypothesize that Zika virus antibodies will exhibit the broadest protection if they bind to functionally conserved domains of the Zika virus envelope (E) glycoprotein. The goal of this project is to define the capacity of the ZIKV E protein to tolerate amino acid substitutions. We will do so by generating libraries of ZIKV where each amino acid of the E protein is changed one at a time to all other possible amino acids. Deep sequencing of these libraries before and after passaging in cell culture will allow the determination of the relative fitness of each mutant. We will also examine how E protein mutations impact antibody mediated neutralization by passaging E protein mutant libraries in the presence of neutralizing antibodies. These studies will both precisely map the epitope of each antibody and define the capacity of ZIKV to evolve resistance to antibody inhibition. This approach can be easily adapted to study ZIKV E functions in a diverse range of cell culture and in vivo scenarios, and define functional determinants of any E protein-directed antibodies that negatively impact infection. The results of this project will not only aid in the development of therapeutic antibodies and vaccination efforts for Zika virus, but can be further applied in combating other virus outbreaks.